DNA-binding proteins are proteins that comprise any of many DNA-binding domains and thus have a specific or general affinity to DNA. DNA-binding proteins include for example transcription factors that modulate the process of transcription, nucleases that cleave DNA molecules, and histones that are involved in DNA packaging in the cell nucleus.
Transcription factors are usually defined as proteins that show sequence-specific DNA binding affinity and that are capable of activating and/or repressing transcription. The Arabidopsis thaliana genome codes for at least 1533 transcriptional regulators, accounting for ˜5.9% of its estimated total number of genes (Riechmann et al. (2000) Science 290: 2105-2109). The Database of Rice Transcription Factors (DRTF) is a collection of known and predicted transcription factors of Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica, and currently contains 2,025 putative transcription factors (TF) gene models in indica and 2,384 in japonica, distributed in 63 families (Gao et al. (2006) Bioinformatics 2006, 22(10):1286-7).
One of these families is the Growth-Regulating Factor (GRF) family of transcription factors, which is specific to plants. At least nine GRF polypeptides have been identified in Arabidopsis thaliana (Kim et al. (2003) Plant J 36: 94-104), and at least twelve in Oryza sativa (Choi et al. (2004) Plant Cell Physiol 45(7): 897-904). The GRF polypeptides are characterized by the presence in their N-terminal half of at least two highly conserved domains, named after the most conserved amino acids within each domain: (i) a QLQ domain (InterPro accession IPR014978, PFAM accession PF08880), where the most conserved amino acids of the domain are Gln-Leu-Gln; and (ii) a WRC domain (InterPro accession IPR014977, PFAM accession PF08879), where the most conserved amino acids of the domain are Trp-Arg-Cys. The WRC domain further contains two distinctive structural features, namely, the WRC domain is enriched in basic amino acids Lys and Arg, and further comprises three Cys and one His residues in a conserved spacing (CX9CX10CX2H), designated as the Effector of Transcription (ET) domain (Ellerstrom et al. (2005) Plant Molec Biol 59: 663-681). The conserved spacing of cysteine and histidine residues in the ET domain is reminiscent of zinc finger (zinc-binding) proteins. In addition, a nuclear localisation signal (NLS) is usually comprised in the GRF polypeptide sequences.
Interaction of some GRF polypeptides with a small family of transcriptional coactivators, GRF-interacting factors (GIF1 to GIF3; also called synovial sarcoma translocation (SYT) polypeptide, SYT1 to SYT3), has been demonstrated using a yeast two-hybrid interaction assay (Kim & Kende (2004) Proc Natl Acad Sci 101: 13374-13379).
The name GRF has also been given to another type of polypeptides, belonging to the 14-3-3 family of polypeptides (de Vetten & Ferl (1994) Plant Physiol 106: 1593-1604), that are totally unrelated the GRF polypeptides useful in performing the methods of the invention.
Transgenic Arabidopsis thaliana plants transformed with a rice GRF (OsGRF1) polypeptide under the control of a viral constitutive 35S CaMV promoter displayed curly leaves, severely reduced elongation of the primary inflorescence, and delayed bolting (van der Knapp et al. (2000) Plant Physiol 122: 695-704). Transgenic Arabidopsis thaliana plants transformed with either one of two Arabidopsis GRF polypeptides (AtGRF1 and AtGRF2) developed larger leaves and cotyledons, were delayed in bolting, and were partially sterile (due to lack of viable pollen), compared to wild type plants (Kim et al. (2003) Plant J 36: 94-104).
In US patent application US2006/0048240, an Arabidopsis thaliana GRF polypeptide is identified as SEQ ID NO: 33421. In US patent application US 2007/0022495, an Arabidopsis thaliana GRF polypeptide is identified as SEQ ID NO: 1803 (also therein referred to as G1438). Transgenic Arabidopsis plants overexpressing G1438 using the 35S CaMV promoter present dark green leaves.
Surprisingly, it has now been found that increasing expression of a nucleic acid sequence encoding a GRF polypeptide gives plants having increased yield-related traits relative to control plants.
According to one embodiment, there is provided a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression of a nucleic acid sequence encoding a GRF polypeptide in a plant. The increased yield-related traits comprise one or more of: increased early vigour, increased aboveground biomass, increased total seed yield per plant, increased seed filling rate, increased harvest index and increased thousand kernel weight.
Little is known about the molecular biology of root formation in monocotyledonous plants. So far only a few genes have been identified that affect root development: examples are the rt1 mutant which forms few or no crowns and brace roots (Jenkins, J. Hered. 21: 79-80, 1930), the asr1 mutant, which displays defective seminal roots (De Miranda et al., Maize Genet. Coop. News Lett. 54: 18-19, 1980), the rtcs mutant lacking nodal (adventitious) roots (Hetz et al., Plant J. 10: 845-857, 1996), the slr1 mutant and slr2 mutant with shortened lateral roots (Hochholdinger et al., Plant Physiol 125:1529-1539, 2001), or rum1, which is affected in lateral initiation in the primary root but also in the initiation of seminal root formation (Woll et al., Plant Physiol., 139, 1255-1267, 2005). Liu et al. (Proteomics 6, 4300-4308, 2006) made a proteomic comparison between primary roots of wild-type and rum1 seedlings and identified another 12 genes that were differently regulated and which were involved in lignin biosynthesis, defence, and the citrate cycle.
Another gene involved in root formation in monocotyledonous plants is raa1, first isolated from rice (Ge et al., Plant Physiol. 135, 1502-1513, 2004): the gene encodes a 12.0-kD protein having 58% homology to the Arabidopsis FPF1 (Flowering Promoting Factor 1). In rice, RAA1 was expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive overexpression increased the number of adventitious roots, but primary root growth was decreased. In addition, the endogenous auxin content was increased. OsRAA1 was also induced by auxin; suggesting that a positive feedback regulation exists between RAA1 and auxin in rice root development (Ge et al., 2004). Furthermore, plants overexpressing OsRAA1 had longer leaves and sterile florets (Ge et al., 2004). WO 2006/067219 discloses the use of FPF1 and related proteins for increasing the production of carbohydrates in plants, but transgenics overexpressing FPF1 did not show increased seed yield and no effects on root growth were reported.
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a RAA1-like polypeptide gives plants having enhanced yield-related traits, in particular increased yield relative to control plants.
According one embodiment, there is provided a method for improving yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding a RAA1-like polypeptide in a plant. The improved yield related traits comprised increased height, shoot/root index, root thickness, greenness index, number of flowers per panicle and increased thousand kernel weight. Improved yield related traits were observed under normal growth conditions as well as under stress conditions.
Seed Yield Regulator (SYR) is a new protein that hitherto has not been characterised. SYR shows some homology (around 48% sequence identity on DNA level, around 45% at protein level) to an Arabidopsis protein named ARGOS (Hu et al., Plant Cell 15, 1951-1961, 2003; US 2005/0108793). Hu et al. postulated that ARGOS is a protein of unique function and is encoded by a single gene. The major phenotypes of ARGOS overexpression in Arabidopsis are increased leafy biomass and delayed flowering. In contrast, overexpression of SYR in rice primarily increases seed yield, whereas the leafy biomass and flowering time are not obviously affected.
Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a Seed Yield Regulator protein (hereafter named SYR) gives plants, when grown under abiotic stress conditions, having enhanced abiotic stress tolerance relative to control plants.
Therefore, the present invention provides a method for enhancing yield-related traits in plants grown under abiotic stress conditions, relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a SYR polypeptide.
ARKL polypeptides comprise a RING finger domain which resembles that found in the mouse protein ARKADIA, an E3 ubiquitin ligase involved in Nodal signaling during embryogenesis (Mavrakis et al. 2007; PLoS Biol. 2007 March; 5(3):e67).
Ubiquitilytion, a process by which a protein is modified the by covalent attachment of ubiquitin is a central and essential part of various cellular processes in eukaryotes. In plants, defects in this pathway cause numerous development aberrations, altered response to external stimuli and modify cell cycle and growth patterns. Ubiquitinated proteins are targeted for degradation via a 26S proteasome dependent or independent pathway. Ubiquitin modification plays a role in activation of signalling proteins, endocytosis, sorting, and histone modification.
The fate of the ubiquitinated protein is determined by the nature of the ubiquitin linkage. Single or multiple ubiquitins may be attached to the target (mono and poly ubiquitination; the specific Lys residue used to form the ubiquitin chain can influence the final fate of the modified protein, for example whether that is degradation or activation
The attachment of ubiquitin to proteins occurs in a multistep process involving three enzymes called, E1, E2, E3 (Glikcman and Ciechanover (2000) Physiol Rev 82: 377-482). Initially the ubiquin is linked to protein in an ATP dependent manner which is then transferred to a cystein acceptor in the E2 protein to form a E2-ubiquitin intermediate which acts as a ubiquitin donor to the target protein in a reaction mediated by the ubiquitin ligase, also called E3 ligase or E3 enzyme. There are multiple types of E3 ligases. The RING-type E3 ligases are characterized by the presence of a conserved protein domain called RING finger or RING-ZnF (Really Interesting New Gene-Zinc Finger).
Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. Most ZnF proteins contain multiple finger-like protrusions that make tandem contacts with their target molecule, often recognising extended substrates. The RING finger is a specialized Zinc biding domain which peresumably functions in protein—protein interactions. The RING finger is 40 to 60 residues long and coordinates two zinc atoms. It is distinct from other zinc fingers in that the eight metal ligand amino acid residues that coordinate the zinc ion fall into a specific structure called the cross-brace structure (Borden (2000). J Mol Biol 295: 1103-1112). The spacing of the cysteines/histidines coordinating the Zinc ions in such a domain is C-x(2)-C-x(9 to 39)-C-x(1 to 3)-H-x(2 to 3)-C-x(2)-C-x(4 to 48)-C-x(2)-C. Metal ligand pairs one and three co-ordinate to bind one zinc ion, whilst pairs two and four bind the second. There are two different variants, the C3HC4-type and a C3H2C3-type, which is clearly related despite the different cysteine/histidine pattern. The latter type is sometimes referred to as ‘RING-H2 finger’. In the latter the coordination of the Zinc ion is mediated by 6 cysteins and 2 histidines whilst in the C3HC4 is mediated by 7 cysteins and one histidine.
In Arabidopsis thaliana there are at least 477 putative RING domain comprising proteins. Some contain multiple RING finger domains. The RING domains have been classified into eight types based on of the metal ligand residue present and/or the number of amino acids between them (Stone at al. 2005) Plant Phys. 137, 13-30. The RING-H2 class is the largest class in Arabidopsis. Based on the nature of the domains and their organisation the Arabidopsis RING finger proteins have been further classified in 30 groups, Group 1 to Group 30. Subgroups within some of the groups were also recognized, eg. subgroup 2.1 and 2.2 of group 2 (Stone et al. 2005). Group I was referred to as group of RING finger protein lacking previously described domains. Sequence analysis of those protein revealed regions of similarity between a few proteins outside of the RING domain, which were called DAR1 to DAR3 (Domain Associated with RING). DAR1 and DAR3 are approximately 40 amino acids long and DAR2 120. DAR1 was reported to occur only in proteins of plant origin (Stone et al. 2005). The presence of common conserved domains suggested a related function for the proteins comprising the domains.
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding an ARKL polypeptide gives plants having enhanced yield-related traits in particular increased yield relative to control plants.
According one embodiment, there is provided a method for improving or enhancing yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding an ARKL polypeptide in a plant.
All eukaryotic cells contain elaborate systems of internal membranes which set up various membrane-enclosed compartments within the cell. The endomembrane system is collection of membranous structures involved in transport within the cell. The main components of the endomembrane system are the endoplasmic reticulum, Golgi bodies, vesicles, cell membrane and nuclear envelope. Members of the endomembrane system pass materials through each other or though the use of vesicles. A universal feature of all cells is an outer limiting membrane called the plasma membrane.
Cell membranes are built from lipids and proteins. The association of proteins to the membrane may be via a covalent bond, by which the protein is attached to the lipids of the membrane. In the case of the so called transmembrane proteins, polypeptide chains of the protein actually traverse the lipid bilayer. Association to the membrane may also occur via association of the protein, so called peripherial protein, by non-covalent bonds to the protruding portions of integral membrane proteins.
Transmembrane proteins (TM proteins) have an amphiphilic nature with hydrophobic TM segments (TMSs) and hydrophilic loops. In transmembrane proteins, the portion within the lipid bilayer consists primarily of hydrophobic amino acids. These are usually arranged in an alpha helix so that the polar carboxi (—C═O) and amino (—NH) groups at the peptide bonds can interact with each other rather than with their hydrophobic surroundings. Those portions of the polypeptide that project out from the bilayer tend to have a high percentage of hydrophilic amino acids. Furthermore, those that project into the extracellular space are usually glycosilated.
Transmembrane topology of a protein has been determined based on experimental X-ray crystallography, NMR, gene fusion technique, substituted cysteine accessibility method, Asp(N)-linked glycosylation experiment and other biochemical methods. In addition a number transmembrane topology prediction methods have been developed to determine the structure and function of TM proteins from their amino acid sequences (Möller et al., 2001; Ikeda et al., 2002; Chen et al., 2002).
The analysis of protein sequence similarity between proteins has benefited from developments in the genomics field. A number of domains conserved amongst two or more proteins for which no function has yet been assigned can be carried out using specific algorithms. One such conserved domain is the so called DUF221 domain (Domain of Unknown Function 221) as described in Pfam (Finn et al. Nucleic Acids Research (2006) Database Issue 34:D247-D251). This domain is found in a family of hypothetical transmembrane proteins, none of which have any known function, the aligned region is at 538 residues at maximum length. The domain occurs in a number of proteins of eukaryotic origin. Expression of an Arabidopsis gene, EDR4, encoding a protein comprising a DUF221 has been reported to be expressed shortly upon dehydration treatment (Kiyosue et al; Plant Mol Biol. 1994 25(5):791-8). An Arabidopsis knockout mutant, gfs10, in a gene encoding another DUF221 domain-containing protein has been reported to have a phenotype similar to that of vacuolar sorting mutants (Fuji et al; 2007. Plant Cell. 2007. 19(2):597-609).
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a YTP polypeptide gives plants having enhanced yield-related traits in particular increased yield relative to control plants.
According one embodiment, there is provided a method for enhancing (improving) yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding a YTP polypeptide in a plant.